Application of mecobalamine in preparation of preparation for improving imbalance of intestinal flora
By using methylcobalamin in the preparation to regulate the intestinal flora, the problem of intestinal flora imbalance caused by a high-fat diet is solved, and the effect of improving the intestinal flora structure and preventing related diseases is achieved.
Patent Information
- Application Number
- CN202411341490.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-25
- Publication Date
- 2025-02-14
AI Technical Summary
A high-fat diet leads to imbalance in intestinal flora, and the occurrence and development of related diseases such as obesity, diabetes and inflammatory bowel disease are related to the development. There is no effective regulatory method in the existing technology.
In preparations for improving intestinal flora imbalance using methylcobalamin or its pharmaceutically acceptable salts, polymorphs, eutectics, solvates, stable isotope derivatives, metabolites or prodrugs .
Methylcobalamin can regulate the alpha diversity, Beta diversity, phylum level and family level of intestinal flora, improve intestinal flora imbalance, and has developed to improve the intestinal flora imbalance of subjects or treat and prevent-related diseases. potential.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to application of methylcobalamin in preparing a preparation for improving intestinal flora imbalance. Background Art
[0002] The intestinal flora and the host are in a state of mutual symbiosis and dynamic equilibrium. The intestinal flora is mainly composed of anaerobic bacteria, facultative anaerobic bacteria and aerobic bacteria. It produces a variety of vitamins and enzymes required by the body, participates in the digestion and metabolism of nutrients, and also produces antimicrobial peptides such as defensins, C-type lectins, and antimicrobial peptides, which protect the body from pathogen invasion and play a vital role in maintaining the health of the host. In addition, short-chain fatty acids (SCFA), an important metabolite of the intestinal flora, can act on proinflammatory cytokines, inhibit inflammatory responses, and maintain the integrity and homeostasis of the intestinal mucosa. Genetics, environment, diet, disease, antibiotics and other factors can all lead to intestinal flora imbalance, which is manifested as changes in the composition and structure of the intestinal flora and abnormal diversity; pathogenic and conditionally pathogenic microorganisms dominate; intestinal barrier dysfunction, increased intestinal wall permeability, bacterial translocation into the systemic circulation, and then induce inflammatory responses and metabolic disorders in the body.
[0003] The proportion of high-fat diet in people's daily diet is gradually increasing. Long-term intake of high-fat food will cause a variety of pathological changes, such as hyperinsulinemia, insulin resistance, dyslipidemia, overstimulation of the sympathetic nervous system, systemic inflammation, hyperglycemia, endotoxemia, increased reactive oxygen species and oxidative stress. A high-fat diet will change the composition of the intestinal microbiota. Studies have shown that a high-fat diet will reduce the number of beneficial bacteria in the intestines, such as Bifidobacterium and Lactobacillus, while increasing potentially harmful bacteria, such as sulfate-reducing bacteria (Desulfovibrio), or causing abnormal changes in the composition ratio of the original microbiota. This change in the structure of the microbial community may be related to the development of chronic diseases such as obesity, diabetes, and inflammatory bowel disease. Secondly, a high-fat diet may also promote cancer progression through metabolic disorders caused by intestinal microbiota, such as increasing the number of Desulfovibrio in the intestine, promoting the progression or recurrence of malignant tumors such as breast cancer and melanoma. In addition, a high-fat diet may also affect the metabolism and health of the host by affecting the metabolites of the intestinal microbiota. For example, a high-fat diet reduces the production of short-chain fatty acids, which have anti-inflammatory effects and may help improve diabetes and obesity. Therefore, it is of great significance to provide a substance that can regulate the intestinal flora imbalance caused by a high-fat diet.
[0004] Methylcobalamin, chemical name, cobalt-α-[α-5,6-dimethylbenzimidazole]-cobalt-β-methylcobalaminamide, also known as: methylcobalamin; methylcobalamin; methylcobalamin; methyl vitamin B, etc. The molecular formula of this compound is C 63 H 90 CoN 13 O 14 P, molecular weight: 1343.4, CAS number: 13422-55-4. Methylcobalamin is an endogenous coenzyme B12 that participates in the one-carbon unit cycle and plays an important role in the transmethylation reaction of homocysteine to methionine. It is easier to enter neuronal organelles than cyanocobalamin. The main pharmacological effects reported in existing literature include direct entry into nerve cells and stimulating the regeneration of damaged axonal areas; promoting the metabolism of nucleic acids, proteins and fats in nerve tissues, stimulating the synthesis of Schwann cell lecithin, repairing damaged myelin sheaths, and improving nerve conduction velocity; stimulating protein synthesis in nerve cells, strengthening the anabolism of axons, and preventing axonal degeneration; participating in nucleic acid synthesis and promoting hematopoietic function. So far, there has been no report on the role of methylcobalamin in regulating the imbalance of intestinal flora caused by a high-fat diet. Summary of the invention
[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention aims to provide the use of methylcobalamin in the preparation of a preparation for improving intestinal flora imbalance.
[0006] In order to achieve the above object, the technical solution adopted by the present invention is:
[0007] The present invention provides use of methylcobalamin or a pharmaceutically acceptable salt, polymorph, cocrystal, solvate, stable isotope derivative, metabolite or prodrug thereof in the preparation of a preparation for improving intestinal flora imbalance in a subject, or treating and / or preventing a disease related to intestinal flora imbalance in a subject.
[0008] In one or more embodiments, the subject has at least one of the following characteristics:
[0009] (1) The subjects' intestinal flora imbalance was caused by a high-fat diet;
[0010] (2) The observed_otus, Chao1 index, Shannon index, and Pielou-e index of the subjects' intestinal flora alpha diversity index decreased;
[0011] (3) The abundance of Bacteroidetes, Verrucomicrobiota, and Desulfobacterota in the subjects' intestines was higher than that in the normal population; the abundance of Firmicutes in the subjects' intestines was lower than that in the normal population;
[0012] (4) The abundance of Akkermansiaceae, Desulfovibrionaceae, and Prevotellaceae in the subjects' intestines was higher than that in the normal population; the abundance of Lachnospiraceae and Muribaculaceae in the subjects' intestines was lower than that in the normal population;
[0013] (5) The abundance of Verrucomicrobiota, Verrucomicrobiae, and Akkermansiaceae in the subjects' intestines was higher than that in the normal population; the abundance of Firmicutes, Clostridia, and Lachnospirales in the subjects' intestines was lower than that in the normal population;
[0014] The normal group refers to a healthy group that does not follow a high-fat diet.
[0015] In one or more embodiments, improving the imbalance of intestinal flora in the subject comprises at least one of the following:
[0016] (1) Improve the observed_otus, Chao1 index, Shannon index, and Pielou-e index in the subjects' intestinal flora alpha diversity index;
[0017] (2) increase the abundance of Firmicutes in the intestinal flora of the subjects and decrease the abundance of Verrucomicrobia in the intestinal flora of the subjects;
[0018] (3) increase the abundance of Lachnospiraceae in the intestinal flora of the subjects and decrease the abundance of Akkermansiaceae in the intestinal flora of the subjects;
[0019] (4) The abundance of Olsenella and Atopobiaceae in the intestinal flora of the subjects was increased, and the abundance of Alloprevotella and Prevotella in the intestinal flora of the subjects was decreased.
[0020] In one or more embodiments, the related diseases include at least one of abnormal glucose and lipid metabolism diseases, gastrointestinal diseases, cardiovascular diseases, and mental and nervous system diseases.
[0021] In one or more embodiments, the abnormal glucose and lipid metabolism disease comprises at least one of obesity, obesity complications, diabetes, and diabetic complications.
[0022] In one or more embodiments, the gastrointestinal disease comprises at least one of inflammatory bowel disease, irritable bowel syndrome, and celiac disease.
[0023] In one or more embodiments, the cardiovascular disease comprises at least one of coronary heart disease, heart failure, hypertension, atrial fibrillation, acute myocardial infarction, and coarctation of the aorta.
[0024] In one or more embodiments, the psychiatric and neurological disorders comprise at least one of anxiety, depression, epilepsy, ALS, autism, or progeria.
[0025] In one or more embodiments, the preparation is any one of a medicine, a health product, a special medical food, a health functional food, a beverage and a feed.
[0026] In one or more embodiments, the preparation further comprises at least one acceptable excipient.
[0027] In one or more embodiments, the formulation is a pharmaceutical product.
[0028] In one or more embodiments, the dosage form of the drug is any one of tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.
[0029] In one or more embodiments, the drug is administered orally, intravenously, intraarterially, subcutaneously, intraperitoneally, intramuscularly or transdermally.
[0030] The beneficial effects of the present invention are: the present invention provides a new application of methylcobalamin in regulating intestinal flora imbalance. Methylcobalamin can regulate the intestinal flora in terms of Alpha diversity, Beta diversity, phylum-level mouse intestinal flora structure, family-level mouse intestinal flora structure, etc., and can be developed into a preparation for improving the intestinal flora imbalance of a subject, or treating and / or preventing diseases related to the intestinal flora imbalance of a subject. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 This is the effect of methylcobalamin on blood biochemical parameters of mice fed with a high-fat diet. TG stands for triglycerides, and UA stands for uric acid.
[0032] Figure 2 Beta diversity analysis results of the effect of methylcobalamin on the intestinal flora of mice fed a high-fat diet.
[0033] Figure 3 It is the dominant bacterial flora at the phylum level of the intestinal flora of each group of mice.
[0034] Figure 4 It is the dominant bacterial flora at the family level in the intestinal flora of each group of mice.
[0035] Figure 5 Analyze cladograms for LEfSe.
[0036] Figure 6 It is the LDA distribution histogram based on LEfSe analysis. DETAILED DESCRIPTION
[0037] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.
[0038] The test materials involved in the embodiments of the present invention are as follows:
[0039] 1. Instruments: veterinary fully automatic biochemical analyzer (BS-240VET), normal temperature centrifuge (Eppendorf Centrifuge 5424), refrigerated centrifuge (Microfuge R 22R Centrifuge), vortex shaker (Huali Da WH-861 Vortex Shaker), three-temperature three-control constant temperature water bath (Shanghai Boxun Industrial Co., Ltd. DK-8D), PCR instrument (Hangzhou Langji Scientific Instrument Co., Ltd. A200 gene amplification instrument) electrophoresis instrument (Tanon Tianneng R EPS300) gel imager (Tanon-2500) ultra-low temperature freezing storage box (Zhongke Meiling Cryogenic Technology Co., Ltd. DW-HL388), fully automatic sample rapid grinding instrument (Shanghai Jingxin JXFSTPRP-96L).
[0040] 2. Reagents: Methylcobalamin dispersible tablets (Zhuohe Pharmaceutical), Pusion Hot start flex 2X Master Mix (Shanghai Yitao Biological Instrument Co., Ltd., M0536L), DL2000 DNA Maker (Takara, 3427A), Gene colour (Beijing Jinboyi, GBY-1), Qubit dsDNA HS Assay Kit (500 times) (Invitrogen, Life technologies, Q3285)4, Biowest Agarose G-10 (BIOWEST, 111860), 50xTAE Buffer (Shanghai Shenggong, B548101-500), AMPure XT beads (Beckman, A63880).
[0041] 3. Animals: 30 SPF-grade C57BL / 6 female mice, 7 weeks old, weighing about 18-20 grams.
[0042] 4. Feed: high-fat feed (Jiangsu Medison Biotechnology Co., Ltd., 60% fat, MD12033).
[0043] Example 1 Effect of methylcobalamin on high-fat diet mice
[0044] Experimental methods
[0045] The mice in the normal group were fed with ordinary feed, and the mice in the model group and the treatment group were fed with high-fat feed. At the 14th week of feeding, the mice in the treatment group were gavaged with 5% Tween-80 aqueous solution (0.308 mg / kg) of methylcobalamin daily, and the mice in the normal group and the model group were gavaged with an equal volume of 5% Tween-80 aqueous solution for 4 weeks. After the last administration, the mice in each group were fasted but not watered for 12 hours, blood was collected to measure biochemical indicators (TG, UA), and the contents of the ileocecal part were collected, and the intestinal flora of each group of mice was detected by 16SrRNA sequencing.
[0046] Experimental Results
[0047] 1. Effects of methylcobalamin on body weight and blood biochemical indexes of mice fed a high-fat diet
[0048] from Figure 1 The results showed that compared with the control (Con) group, the TG index of the model (Mod) group was significantly increased (P<0.001) ( Figure 1 In middle A), the UA index was significantly increased (P<0.01) ( Figure 1 (middle B).
[0049] Compared with the model (Mod) group, the TG index of the mice in the methylcobalamin (Jtreat) group was significantly lower (P<0.001) ( Figure 1 A), the UA index was significantly reduced (P<0.01) ( Figure 1 (middle B).
[0050] 2. Effect of methylcobalamin on intestinal flora of mice fed a high-fat diet
[0051] After DNA extraction from stool samples, the variable region (V3-V4) was amplified using upstream primer 341F (5′-CCTACGGGNGGCWGCAG-3′, SEQ ID NO. 1) and downstream primer 805R (5′-GACTACHVGGGTATCTAATCC-3′, SEQ ID NO. 2). After purification, the amplified DNA was evaluated using an Agilent 2100 bioanalyzer (Agilent, USA) and an Illumina (Kapa Biosciences, Woburn, MA, USA) library quantification kit. Qualified amplified products were sequenced at 2×250 bp using a NovaSeq6000 sequencer and clustered according to a similarity of 97% to obtain operational taxonomic units (OTUs).
[0052] Alpha diversity refers to the diversity within a specific environment or ecosystem, which is mainly used to reflect species richness and uniformity as well as sequencing depth, and is mainly reflected by Chao1, observed_otus, Goods_coverage, Shannon, Simpson and Pielou-e indices. OTUs refers to operational taxonomic units.
[0053] According to the results in Table 1.1 and Table 1.2, the Goods-coverage index of each group of samples was greater than 0.99, indicating that the sequencing results were good and could reflect the microbial community of each sample; compared with the control (Con) group, the observed_otus and Chao1 indices of mice in the model (Mod) group were significantly reduced (P<0.05), and the Shannon and Pielou-e indices were extremely significantly reduced (P<0.001).
[0054] Compared with the mice in the model (Mod) group, the Shannon index of the mice in the methylcobalamin group (Jtreat) was significantly increased (P<0.05), and it had a certain regulatory effect on other indices, making them close to the various indices of Alpha diversity of the intestinal flora of the mice in the control (Con) group.
[0055] Table 1.1: Alpha diversity analysis results of intestinal flora of mice in each group
[0056]
[0057] Note: Data represent (x±SEM), "*" indicates P<0.05 compared with the control group; "***" indicates P<0.001 compared with the control group; "#" indicates P<0.05 compared with the model group.
[0058] Table 1.2: Alpha diversity analysis results of intestinal flora of mice in each group
[0059]
[0060] Note: Data represent (x±SEM), "***" indicates P<0.001 compared with the control group.
[0061] Beta diversity refers to the species differences between different environmental communities. In the principal coordinate analysis PCoA diagram and the principal component analysis PCA diagram, each group of samples can be significantly clustered, indicating that the intestinal flora within each group of samples tends to be consistent. The flora structure composition of the Con group and the Mod group was significantly different (P<0.05), indicating that the high-fat diet caused the intestinal flora structure of mice to be disordered; compared with the Mod group, the samples of the Jtreat group were more closely distributed in the PcoA cluster, and deviated from the Mod group and tended to the Con group, indicating that methylcobalamin administration has a regulatory effect on the intestinal flora structure. Results are shown in Figure 2 .
[0062] Changes in the structure of the intestinal flora of mice at the phylum level: The dominant flora at the phylum level of the intestinal flora of each group of mice are as follows Figure 3 As shown. At the phylum level, Firmicutes and Bacteroidetes were dominant in the intestinal flora of the Con group, with a total abundance of more than 80%; compared with the Con group, the abundance of Firmicutes in the Mod group was significantly reduced, and the abundance of Verrucomicrobiota increased; compared with the Mod group, the abundance of Firmicutes in the Jtreat group increased, and the abundance of Verrucomicrobiota decreased, approaching that of the Con group. This indicates that the administration of methylcobalamin restored the relative abundance of Firmicutes and Verrucomicrobiota in the intestinal flora of mice fed a high-fat diet to the level of the intestinal flora structure of healthy mice.
[0063] Changes in the structure of the intestinal flora of mice at the family level: The dominant flora at the family level of the intestinal flora of each group of mice are as follows Figure 4 As shown. Figure 4It can be seen that at the family level, Lachnospiraceae and Muribaculaceae were the main families in the intestinal flora of the Con group, with an abundance of more than 10%; compared with the Con group, the abundance of these two families in the Mod group decreased significantly, while the abundance of Akkermansiaceae increased; compared with the Mod group, the abundance of Akkermansiaceae decreased in the Jtreat group, while the abundance of Lachnospiraceae increased. This indicates that the administration of methylcobalamin restored the relative abundance of Akkermansiaceae and Lachnospiraceae in the intestinal flora of mice fed a high-fat diet to the level of the intestinal flora structure of healthy mice.
[0064] The LEfSe analysis of the evolutionary branch diagram further determined the differential bacterial community information closely related to the regulatory effect of methylcobalamin. Figure 5 It can be seen that the circles radiating from the inside to the outside in the LEfSe evolutionary branch diagram represent the classification level from phylum to genus. Each small circle represents a classification at that classification level, and its diameter is positively correlated with the relative abundance. Species with no significant differences are shown in yellow, and the differential biomarkers of each group in the experiment are shown in red (Con group), green (Mod group) and blue (Jtreat group). Figure 6 The LDA distribution histogram based on LEfSe analysis is shown, indicating the biomarkers with significant differences in abundance (LDA>3, P<0.05) in different groups. The length of the histogram in the figure represents the degree of influence of the biomarkers with significant differences. The results showed that there were 15, 5 and 12 statistically different bacterial families in the Con group, Mod group and Jtreat group, respectively (LDA>3, P<0.05).
[0065] LEfSe analysis results showed that compared with the Con group, the abundance of Verrucomicrobiota, Verrucomicrobiae, and Akkermansiaceae increased in the Mod group, while the abundance of Firmicutes, Clostridia, and Lachnospirales decreased; after methylcobalamin was administered to the intestinal flora of mice fed a high-fat diet, the number and abundance of flora species in the intestine of mice fed a high-fat diet increased significantly. Compared with the Mod group, the abundance of beneficial bacteria such as Olsenella and Atopobiaceae increased in the Jtreat group, while the abundance of Alloprevotella and Prevotella decreased. The increase in the abundance of Olsenella can help the immune system fight cancer tumors more effectively and help improve the effectiveness of immunotherapy. Some members of the family Paramyxozoites can limit the colonization of vancomycin-resistant enterococci in the mouse intestine by consuming fructose, which helps prevent the colonization of pathogens and thus maintain intestinal health. The reduced abundance of Prevotella and Pseudo-Prevotella may be related to the reduction of inflammatory responses.
[0066] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. Use of methylcobalamin or a pharmaceutically acceptable salt, polymorph, cocrystal, solvate, stable isotope derivative, metabolite or prodrug thereof in the preparation of a preparation for improving intestinal flora imbalance in a subject, or treating and / or preventing a disease related to intestinal flora imbalance in a subject.
2. The use according to claim 1, characterized in that: The subject has at least one of the following characteristics: (1) The subjects' intestinal flora imbalance was caused by a high-fat diet; (2) The observed_otus, Chao1 index, Shannon index, and Pielou-e index of the subjects' intestinal flora alpha diversity index decreased; (3) The abundance of Bacteroidetes, Verrucomicrobiota, and Desulfobacterota in the subjects' intestines was higher than that in the normal population; the abundance of Firmicutes in the subjects' intestines was lower than that in the normal population; (4) The abundance of Akkermansiaceae, Desulfovibrionaceae, and Prevotellaceae in the subjects' intestines was higher than that in the normal population; the abundance of Lachnospiraceae and Muribaculaceae in the subjects' intestines was lower than that in the normal population; (5) The abundance of Verrucomicrobiota, Verrucomicrobiae, and Akkermansiaceae in the intestines of the subjects was higher than that in the intestines of the normal group; the abundance of Firmicutes, Clostridia, and Lachnospirales in the intestines of the subjects was lower than that in the intestines of the normal group.
3. The use according to claim 1, characterized in that: Improving the imbalance of intestinal flora in the subject includes at least one of the following: (1) Improve the observed_otus, Chao1 index, Shannon index, and Pielou-e index in the subjects' intestinal flora alpha diversity index; (2) increase the abundance of Firmicutes in the intestinal flora of the subjects and decrease the abundance of Verrucomicrobia in the intestinal flora of the subjects; (3) increase the abundance of Lachnospiraceae in the intestinal flora of the subjects and decrease the abundance of Akkermansiaceae in the intestinal flora of the subjects; (4) The abundance of Olsenella and Atopobiaceae in the intestinal flora of the subjects was increased, and the abundance of Alloprevotella and Prevotella in the intestinal flora of the subjects was decreased.
4. The use according to claim 1, characterized in that: The related diseases include at least one of abnormal glucose and lipid metabolism diseases, gastrointestinal diseases, cardiovascular diseases, and mental and nervous system diseases.
5. The use according to claim 4, characterized in that: The abnormal glucose and lipid metabolism disease includes at least one of obesity, obesity complications, diabetes, and diabetic complications; Preferably, the gastrointestinal disease comprises at least one of inflammatory bowel disease, irritable bowel syndrome, and celiac disease; Preferably, the cardiovascular disease comprises at least one of coronary heart disease, heart failure, hypertension, atrial fibrillation, acute myocardial infarction, and aortic coarctation; Preferably, the psychiatric and neurological disorders include at least one of anxiety, depression, epilepsy, ALS, autism or progeria.
6. The use according to claim 1, characterized in that: The preparation is any one of medicines, health products, special medical foods, health functional foods, beverages and feeds.
7. The use according to claim 1, characterized in that: The preparation further comprises at least one acceptable excipient.
8. The use according to claim 1, characterized in that: The preparation is a medicine.
9. The use according to claim 8, characterized in that: The dosage form of the drug is any one of tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.
10. The use according to claim 8, characterized in that: The drug is administered orally, intravenously, intraarterially, subcutaneously, intraperitoneally, intramuscularly or transdermally.